Use of purified 2′-fucosyllactose, 3-fucosyllactose and lactodifucotetraose as prebiotics

ABSTRACT

The invention provides compositions and methods for utilizing synthetic human milk oligosaccharides as prebiotics.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Application No. 61/485,895, filed May 13, 2011,which is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government Support under Contract NumberNIAID U01AI075563 by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention provides compositions and methods for utilizingoligosaccharides as prebiotics, in particular certain fucosylatedoligosaccharides that are typically found in human milk.

BACKGROUND OF THE INVENTION

Human milk contains a diverse and abundant set of neutral and acidicoligosaccharides. Although these molecules may not be utilized directlyfor nutrition, they nevertheless serve critical roles in the preventionof disease, in immune function, and in the establishment of a healthygut microbiome. The bacteria present in the human intestine arecritically associated with human health, and an abnormal/pathogenicintestinal microflora can be detrimental to host survival.

SUMMARY OF THE INVENTION

To maximize the beneficial effect of endogenous commensal or symbioticmicrobes or exogenously-administered probiotics, prebiotic agents areadministered to stimulate the growth and/or activity ofadvantageous/symbiotic bacteria in the digestive system. As such, thereis a pressing need to develop new prebiotic agents that selectivelyimprove the survival and effectiveness of beneficial/symbiotic microbes.The invention is based on the discovery that fucosylated human milkoligosaccharides in a highly purified form, e.g., 2′-fucosyllactose(2′-FL), 3-fucosyllactose (3-FL), or lactodifucotetraose (LDFT), areuseful as prebiotic compositions. The prebiotic compositions describedherein are indigestible or partially indigestible (by the host), butprovide health benefits to the host by promoting the growth of certainprobiotic microorganisms. The compositions preferentially promote growthof probiotic bacteria compared to pathogenic microorganisms. Thus, theinvention provides prebiotic compositions comprising purified human milkoligosaccharides (HMOS) in an amount effective to stimulate the growthof endogenous commensal or symbiotic microbes or exogenouslyadministered probiotics. Preferably, the oligosaccharide compositioncomprises purified 2′-fucosyllactose (2′-FL), purified 3-fucosyllactose(3-FL), purified lactodifucotetraose (LDFT), or combinations thereof. Acombination of purified 2′-FL and purified 3-FL synergisticallystimulates the growth and/or activity of advantageous/symbiotic bacteriain the digestive system. Optionally, purified LDFT is added to thiscombination. Preferably, the 2′-FL, 3-FL, or LDFT is 95-99% pure. Theprebiotic oligosaccharides of the invention are administered alone(i.e., in the absence of exogenously administered bacteria).Alternatively, the prebiotic oligosaccharides of the invention areadministered in conjunction with live microorganisms (i.e., probiotics).The composition optionally includes a pharmaceutically-acceptableexcipient or inactive ingredients.

As used herein, an “isolated” or “purified” oligosaccharide issubstantially free of other oligosaccharides, with which it naturallyoccurs in human milk. Purified oligosaccharides are also free ofcellular material when produced biosynthetically, or other chemicalswhen chemically synthesized. Purified compounds are at least 60% (by dryweight) of the compound of interest. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight the compound of interest. For example, a purifiedoligosaccharide, e.g., 2′-FL, 3-FL, or LDFT is one that is at least 90%,91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desiredoligosaccharide by weight. Purity is measured by any appropriatestandard method, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis. The oligosaccharides are purified and used in a number ofproducts for consumption by humans as well as animals, such as companionanimals (dogs, cats) as well as livestock (bovine, equine, ovine,caprine, or porcine animals, as well as poultry). “Purified” alsodefines a degree of sterility that is safe for administration to a humansubject, e.g., lacking infectious or toxic agents.

Similarly, by “substantially pure” is meant an oligosaccharide that hasbeen separated from the components that naturally accompany it.Typically, the oligosaccharide is substantially pure when it is at least60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteinsand naturally-occurring organic molecules with which it is nanturallyassociated.

The prebiotic compositions described herein stimulate the growth ofendogenous commensal or symbiotic microbes or exogenously administeredprobiotics of various genera including Bifidobacterium, Lactobacillus,or Streptococcus. Saccharomyces boulardii is a suitable probiotic yeast.Suitable probiotic bacteria include Bacillus coagulans (e.g. Bacilluscoagulans GBI-30 or 6086), Bacteroides fragilis, Bacteroidesthetaiotaomicron, Bifidobacterium animalis (e.g. Bifidobacteriumanimalis BB-12), Bifidobacterium breve, Bifidobacterium bifidum (e.g.Bifidobacterium bifidum BB-12), Bifidobacterium infantis (e.g.Bifidobacterium infantis 35624), Bifidobacterium lactis, Bifidobacteriumlongum, Escherichia coli, Enterococcus faecalis, Enterococcus faecium,Lactobacillus acidophilus (e.g. Lactobacillus acidophilus RC-14, NCFM orCL 1285), Lactobacillus casei (e.g. Lactobacillus casei LBC80R),Lactobacillus fermentum (e.g. Lactobacillus fermentum B-54),Lactobacillus paracasei (e.g. Lactobacillus paracasei NCC2461 or8700:2), Lactobacillus johnsonii (e.g. Lactobacillus johnsonii NCC533),Lactococcus lactis, Lactobacillus plantarum (e.g. Lactobacillusplantarum 299v or HEAL 9), Lactobacillus reuteri (e.g. Lactobacillusreuteri SD2112 or DSM 17938), Lactobacillus rhamnosus (e.g.Lactobacillus rhamnosus GG or GR-1), and Streptococcus thermophilus.Preferably, the probiotic bacteria are selected from the groupconsisting of Bifidobacterium bifidum, Bacteroides fragilis, Bacteroidesthetaiotaomicron, Bifidobacterium infantis, Bifidobacterium longum,Lactobacillus acidophilus, Lactobacillus rhamnosus and Streptococcusthermophilus.

The prebiotic composition comprises between 0.01 g oligosaccharide,e.g., 2′-FL, 3-FL, or LDFT, and 10 g oligosaccharide per 10 grams ofcomposition. For example, the prebiotic composition comprises between0.1 g and 5 g 2′-FL, between 0.5 g and 5 g 2′-FL, between 1 g and 5 g2′-FL, or between 1.5 g and 3 g 2′-FL per 10 grams of composition.Alternatively, the 2′-FL comprises 90% to 100% by weight of thecomposition, e.g., 92% to 100%, 95% to 100%, 96% to 100%, 97% to 100%,98% to 100%, or 99% to 100% by weight of the composition.

The mean concentrations of 2′-FL, 3-FL, and LDFT in human milk are asfollows: 2′-FL=2.43 g/L (±0.26), 3-FL=0.86 g/L (±0.10), and LDFT=0.43g/L (±0.04; Chaturvedi P, et al. 2001 Glycobiology, May; 11(5):365-72).Thus, the mean ratio of 2′-FL:3FL:LDFT in human milk is 5.65:2:1. Theprebiotics of the invention are administered in any ratio of2′-FL:3FL:LDFT, e.g., 1:1:1, 5:2:1, 10:5:1, 1:2:5, 1:5:10, 5:1:2,10:1:5, 2:1:5, or 5:1:10, or any other ratio suitable to obtainprebiotic effects. Alternatively, 2′-FL and 3-FL are administeredtogether in the following exemplary ratios of 2′-FL:3-FL 1:1, 1:2, 1:5,1:10, 1:100, 100:1, 10:1, 5:1, or 2:1.

The composition is in the form of a tablet, a capsule, a powder, abeverage, or an infant formula. For example, the purified prebioticoligosaccharides of the invention are in the form of powdered/dry milk.By “infant” is meant a child under the age of 12 months. By “infantformula” is meant a foodstuff intended for particular nutritional use byinfants aged under twelve months and constituting the principal liquidelement in the progressively diversified diet of this category ofperson. Alternatively, the composition is provided in mashed rice, in abanana, in a porridge, or in a gruel. Due to the surge in disease ininfants at weaning, the HMOS of the invention are added to weaning foods(e.g., mashed rice, bananas, porridges and other gruels, formula, etc.)to reduce or ameliorate these diseases. Optionally, the HMOS are addedto the weaning foods during the manufacturing process. Alternatively,the synthetic HMOS are added to the weaning foods after themanufacturing process, but prior to ingestion. For example, packets ofsugars including one or more purified HMOS are added to weaning foodsprior to infant ingestion. In one aspect, the composition furthercomprises probiotic bacteria, which are prevented from prematureutilization of the prebiotic component through physical means (e.g.separation by a physical barrier) or through other means (e.g. throughreversible metabolic stasis of the probiotic organism, e.g. as providedby desiccation or refrigeration). In another aspect, the compositionfurther comprises fructo-oligosaccharides (FOS),galacto-oligosaccharides (GOS), or lactulose.

The purified 2′-FL, 3-FL, or LDFT prebiotic oligosaccharide is alsoadded to other consumable products such as yogurt or probiotic beveragesfor consumption by infants, children, and adults. For example, thepurified 2′-FL, 3-FL, or LDFT prebiotic oligosaccharide is added topowdered/dry milk.

Also provided are methods of stimulating the growth of endogenouscommensal or symbiotic microbes or exogenously administered probioticsin the gastrointestinal tract by administering to the gastrointestinaltract of a subject a composition comprising an effective amount ofpurified human milk oligosaccharides (HMOS). Preferably, the HMOScomprises purified 2′-fucosyllactose (2′-FL), purified 3-fucosyllactose(3-FL), purified lactodifucotetraose (LDFT), or a combination thereof. Acombination of purified 2′-FL, purified 3-FL, and purifiedlactodifucotetraose (LDFT) synergistically stimulates the growth and/oractivity of advantageous/symbiotic bacteria in the digestive system.Also provided is the use of purified 2′-FL, purified 3-FL, and/orpurified LDFT in the manufacture of a medicament for stimulating thegrowth of bacteria in the gastrointestinal tract. Preferably, the 2′-FL,3-FL, or LDFT is 95-99% pure. The composition comprises between 0.01 g2′-FL and 10 g 2′-FL per 10 grams of composition, e.g., between 0.1 gand 5 g 2′-FL, between 0.5 g and 5 g 2′-FL, between 1 g and 5 g 2′-FL,or between 1.5 g and 3 g 2′-FL per gram of composition. Alternatively,the 2′-FL comprises 90% to 100% by weight of the composition, e.g., 92%to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, or 99% to100% by weight of the composition.

Administration of the purified prebiotics described herein improves theoverall health of the gastrointestinal tract by influencing many membersof the microbial community. The prebiotics described herein activatesignaling pathways within the intestinal mucosa, inhibit pathogenbinding to mucosal surfaces, and attenuate inflammation of theintestinal mucosa. For example, administration of the purifiedprebiotics of the invention results in a decrease in infection byenteric pathogens, a decrease in diarrhea, a decrease in constipation, areduction in toxic catabolites, a reduction of intestinal cancer, and anenhanced immune response. In one example, the subject is a mammal inneed of treatment, e.g., a subject that has been diagnosed withinfectious diarrhea, acute infectious diarrhea, antibiotic-associateddiarrhea (AAD), traveler's diarrhea (TD), necrotizing enterocolitis(NEC), inflammatory bowel disease, Helicobacter pylori infection,respiratory tract infection, ear, nose, or throat (ENT) infection, or aninfectious complication in surgical and critically ill patients. Themammal can be, e.g., any mammal, e.g., a human, a primate, a mouse, arat, a dog, a cat, a horse, as well as livestock or animals grown forfood consumption, e.g., cattle, sheep, pigs, chickens, and goats. In apreferred embodiment, the mammal is a human.

The local pH in the gastrointestinal tract is decreased followingadministration of the compositions of the invention. Additionally, thelocal lactate or lactic acid concentration in the gastrointestinal tractis increased following administration of the compositions of theinvention. Finally, the growth of pathogenic bacteria in thegastrointestinal tract is inhibited following administration of thecompositions of the invention.

In one example, the endogenous commensal or symbiotic microbes orexogenously administered probiotics stimulated by the compositions ofthe invention are selected from the group consisting of Bifidobacteriumbifidum, Bacteroides fragilis, Bacteroides thetaiotaomicron,Bifidobacterium infantis, Bifidobacterium longum, Lactobacillusacidophilus, Lactobacillus rhamnosus, Bacteroides vulgatus, Lactococcuslactis, and Streptococcus thermophilus. Alternatively, the compositionsof the invention stimulate the probiotic yeast Saccharomyces boulardii.

In one aspect, the administered composition further comprises probioticbacteria selected from the group consisting of Bifidobacterium bifidum,Bacteroides fragilis, Bacteroides thetaiotaom, Bifidobacterium infantis,Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus icronrhamnosus, Bacteroides vulgatus, Lactococcus lactis, and Streptococcusthermophilus. In another aspect, the composition further comprisesfructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), orlactulose. Preferably, the pathogenic bacteria is selected from thegroup consisting of Clostridium histolyticum, Clostridium difficile,Clostridium perfringens, Enterobacter aerogenes, Listeria monocytogenes,Staphylococcus aureus, Salmonella enterica, Yersinia enterocolitica,enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichiacoli (EIEC), enteroaggregative Escherichia coli (EAEC), enterotoxigenicEscherichia coli, enterohemorrhagic Escherichia coli (EHEC), e.g.,Escherichia coli O157:H7, Escherichia coli 0111:H8, Escherichia coli0104:H21, Escherichia coli 026:H11, Escherichia coli O103:H2,Escherichia coli O111:NM, and Escherichia coli 0113:H21.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. The terms “preventing” and“prevention” refer to the administration of an agent or composition to aclinically asymptomatic individual who is susceptible to a particularadverse condition, disorder, or disease, and thus relates to theprevention of the occurrence of symptoms and/or their underlying cause.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component to provide the desired effect. Forexample, by “an effective amount” is meant an amount of anoligosaccharide to increase the proliferation of endogenous commensal orsymbiotic microbes or exogenously administered probiotics or prolong theresidence time of the bacteria in the gastrointestinal tract of asubject who has consumed the bacteria. Ultimately, the attendingphysician or veterinarian decides the appropriate amount and dosageregimen.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, Genbank/NCBI accession numbers, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of bar charts demonstrating that 2′-fucosyllactose(2′-FL) and human milk oligosaccharides (HMO) induce bacterial growth ininfant microbiota cultures from three healthy babies, identified as A,B, and C.

FIG. 2 is a bar chart demonstrating that 2′-FL and HMO decrease pH ininfant microbiota cultures from three healthy babies, identified as A,B, and C.

FIG. 3 is a series of bar charts demonstrating that 2′-FL and HMOincrease lactate concentration in infant microbiota cultures from threehealthy babies, identified as A, B, and C.

FIG. 4 is a series of bar charts demonstrating the effect of 2′-FL,human milk oligosaccharides (HMOS), and fructo-oligosaccharides (FOS) onthe growth (optical density (OD)) and final culture pH of variousbacteria.

FIG. 5 is a series of bar charts demonstrating the effect of 3-FL, humanmilk oligosaccharides (HMOS), and fructo-oligosaccharides (FOS) on thegrowth (optical density (OD)) and final culture pH of various bacteria.

FIG. 6 is a series of bar charts demonstrating the effect of LDFT, humanmilk oligosaccharides (HMOS), and fructo-oligosaccharides (FOS) on thegrowth (optical density (OD)) and final culture pH of various bacteria.

DETAILED DESCRIPTION

The human intestinal microflora is largely stable when the host is ingood health; however, the ecosystem of the intestinal microflora variesdepending on host age, disease, microorganism infection, stress,nutritional components, and pharmaceutical treatments. Modification ofthe composition and metabolic activity of the intestinal bacterialcommunity, which can confer a health benefit to the host, may beachieved by administration of growth promoting sugars (i.e. prebiotics),by administration of live exogenous microorganisms (i.e., probiotics),or a combination of both.

Described herein are compositions that contain one or more highlypurified prebiotic fucosylated oligosaccharides, e.g., 2′-fucosyllactose(2′-FL), 3-fucosyllactose (3-FL), and lactodifucotetraose (LDFT), inamounts that promote proliferation and residence of endogenous commensalor symbiotic microbes or exogenously administered probiotics in themammalian gut. One or a combination of purified human milkoligosaccharides (HMOS) described herein alter microbiota composition inhuman stools, and stimulate the growth of beneficial/symbiotic bacteriain the human intestine. Certain bacteria present in the human intestineare associated with human health, and an abnormal/pathogenic intestinalmicroflora can be detrimental to host survival. The human intestinalmicroflora is largely stable when the host is in good health; however,the ecosystem of the intestinal microflora may change depending on hostage, disease, microorganism infection, stress, nutritional components,and pharmaceutical administration.

Modification of the composition and metabolic activity of the intestinalbacterial community is performed through the administration of prebioticoligosaccharides alone (i.e., in the absence of exogenously administeredbacteria), beneficial microorganisms (i.e., probiotics), or acombination of prebiotics and probiotics. As described in detail below,to maximize the beneficial effect of endogenous commensal microbes orexogenously administered probiotic microorganisms, prebiotic agents(purified oligosaccharides) are administered to stimulate the growthand/or activity of advantageous bacteria in the digestive system.Described herein are prebiotic agents that selectively improve thesurvival and effectiveness of probiotic microbes. For example, asdescribed in detail below, isolated and/or purified 2′-FL, 3-FL, or LDFTare administered to selectively stimulate the growth of beneficialbacteria.

The Human Microbiome

Mammals appear to be born free of bacteria, fungi, or protozoa; however,exposed organs—skin, body invaginations, and digestive tract—becomeniches for adapted microbes. These mucosal surfaces serve as the body'sinteraction with the external environment. The contents of these organsare kept separated from the “interior” of the body by barriers thateffectively cordon the luminal microbes. Humans have a developmentalprogram for expression of antimicrobial peptides modulating themicrobial ecosystem that begins to form shortly after birth (Menard S,et al. 2008 J Exp Med, 21; 205(1): 183-193). The process of colonizationis dynamic, creating the structured populations reached in the ultimatecommunity, which itself is subject to perturbations (Caufield P W, etal. 1993 J. Dent Res, 72(1):37-45; Schwiertz A, et al. 2003 Pediatr Res,54(3):393-399; Hopkins M J, et al. 2005 FEMS Microbiology Ecology,54(1):77-85). The choreography of such events is modulated by thecomplex interactions involving microbial signaling with other microbesand with the host.

The relationship between gut flora and humans is not merely commensal (anon-harmful coexistence), but rather a symbiotic relationship. Thoughpeople can survive without gut flora, the microorganisms perform avariety of useful functions, such as fermenting unused energysubstrates, training the immune system, preventing growth of pathogenicbacteria, regulating the development of the gut, producing vitamins forthe host (such as biotin and vitamin K), and producing hormones todirect the host to store fats (See, e.g., Dominguez-Bello M G and BlaserM J, 2008 Microbes Infect, 10(9): 1072-1076). However, in certainconditions, pathogenic species are capable of causing disease byproducing infection or increasing cancer risk for the host.

Infant Gut Microbiota

The sterile gastrointestinal tract of the newborn is inoculated withmicroflora from the birth canal, close maternal contact, and theenvironment. Initial colonization is followed by waves of succession.Post-natal development, intrinsically modulated by both genetic andmicrobial factors, leads to a functionally healthy intestine thatmaintains a dynamic balance between microbes, host epithelial cells, andmucosal immunity. The initial pioneering species include facultativeanaerobes such as lactobacilli and bifidobacteria, which initiallyestablish the acidic anaerobic environment in which the increasinglycomplex microbiota is established over time. Exclusive breastfeedingpromotes a human milk-induced intestinal predominance of Bifidobacteriumbifidum (1,000:1 ratio of bifidobacteria to enterobacteria) and otherfavorable strains of bacteria that acidify the gut (stool pH of 5.1 by 7days), which favors further colonization by the same symbiotic anaerobes(bifidobacteria and lactobacilli). A secondary wave of succession isnoted at around 4 months by the increasing presence of Bacteroides spp,including Bacteroides fragilis and Bacteroides thetaiotaomicron, whicheventually become dominant species in the adult microflora.

Probiotic Bacteria

The term “probiotic” is derived from a Greek word meaning “supporting orfavoring life” (See, e.g., Vanderpool C, et al. 2008 Inflamm Bowel Dis,14(11):1585-96). Probiotics are often in the form of dietary supplementscontaining beneficial bacteria or yeasts. The World Health Organizationcharacterizes probiotics as “live microorganisms which when administeredin adequate amounts confer a health benefit on the host” (Report of aJoint FAO/WHO Expert Consultation on Evaluation of Health andNutritional Properties of Probiotics in Food Including Powder Milk withLive Lactic Acid Bacteria (October 2001)). Probiotics are often derivedfrom healthy human microflora, and reduce the risk of disease uponingestion. Common probiotics include Bifidobacterium and Lactobacillusspecies, Streptococcus thermophilus, Enterococcus and Bacillus species,E. coli, and yeasts such as Sacharomyces boulardii. The consumption ofprobiotics reduces diarrhea, necrotizing enterocolitis (NEC),inflammatory bowel diseases, and allergic disease. Probiotics protecthigh-risk infants from NEC by “normalizing” the abnormal bowelcolonization of premature infants by providing an increased barrier totranslocation of bacteria across the gut mucosa, by competitiveexclusion of potential pathogens, and by growth inhibition of bacterialpathogens. Premature infants are poorly colonized by beneficialBifidobacterium, and very low birth weight infants fed probioticcombinations of Bifidobacterium infantis and Lactobacillus acidophilus,or Bifidobacterium infantis, Streptococcus thermophilus, andBifidobacterium bifidus have lower incidence and severity of NEC, andless death, compared with controls.

Probiotics exert direct antibacterial effects on pathogens throughproduction of antibacterial substances, including bacteriocins and acid(Cotter P D, et al. 2005 Nat Rev, 3:777-788; Servin A L, 2004 FEMSMicrobiol Rev, 28: 405-440). These probiotic-derived antibacterialsubstances exert their effects alone or synergistically to inhibit thegrowth of pathogens. Specifically, probiotic bacteria, especiallystrains of lactobacilli, produce acetic, lactic, and propionic acid thatlower the local pH, which inhibits the growth of a wide range ofGram-negative pathogenic bacteria. Probiotics administered to thegastrointestinal tract also decrease adhesion of both pathogens andtheir toxins to the intestinal epithelium. Several strains of beneficialbacteria, including lactobacilli and bifidobacteria are able to competewith pathogenic bacteria, including Clostridium histolyticum,Clostridium difficile, Enterobacter aerogenes, Listeria monocytogenes,Staphylococcus aureus, Salmonella enterica, Yersinia enterocolitica,enterotoxigenic E. coli, and enteropathogenic E. coli for intestinalepithelial cell binding. Moreover, probiotic bacteria displacepathogenic bacteria even if the pathogens have attached to intestinalepithelial cells prior to probiotic administration (Collado M C, et al.2007 Lett Appl Microbiol, 45: 454-460; Candela M, et al. 2005 ResMicrobiol, 156: 887-895). Specific probiotics or probiotic combinationsare selected based on their ability to inhibit or displace a specificpathogen.

Both in vitro and in vivo studies show effects of probiotics on hostimmune functions and intestinal epithelial cell functions (See, e.g.,Vanderpool C, et al. 2008 Inflamm Bowel Dis, 4(11):1585-96).Probiotic-induced upregulation of immune function improves the abilityto fight infections or inhibit tumor formation, while downregulationprevents the onset of allergy or intestinal inflammation. Probioticbacteria stimulate intestinal epithelial cell responses, includingrestitution of damaged epithelial barrier, production of antibacterialsubstances and cell-protective proteins, and blockade ofcytokine-induced intestinal epithelial cell apoptosis. Many of theseresponses result from probiotic stimulation of specific intracellularsignaling pathways in the intestinal epithelial cells.

Human Milk Glycans

Human milk contains a diverse and abundant set of neutral and acidicoligosaccharides. For example, the prebiotic fucosylatedoligosaccharides described herein, e.g., 2′-fucosyllactose (2′-FL),3-fucosyllactose (3-FL), and lactodifucotetraose (LDFT), are typicallyfound in human milk Human milk oligosaccharides as a class survivetransit through the intestine of infants very efficiently, beingessentially indigestible (Chaturvedi, P et al. 2001 Adv Exp Med Biol,501:315-323). Although these molecules may not be utilized directly byinfants for nutrition, they nevertheless serve critical roles in theestablishment of a healthy gut microbiome (Marcobal A, et al. 2010 JAgric Food Chem, 58:5334-5340), in the prevention of disease (Newburg D,et al. 2005 Annu Rev Nutr, 25:37-58), and in immune function (Newburg D,et al. 2007 Pediatr Res, 61: 2-8). Despite millions of years of exposureto human milk oligosaccharides (HMOS), pathogens have yet to developways to circumvent the ability of HMOS to prevent adhesion to targetcells and to inhibit infection. The ability to utilize HMOS as pathogenadherence inhibitors promises to address the current crisis ofburgeoning antibiotic resistance. Human milk oligosaccharides representthe lead compounds of a novel class of therapeutics against some of themost intractable scourges of society.

Human milk glycans, which comprise both unbound oligosaccharides andtheir glycoconjugates, play a significant role in the protection anddevelopment of the infant gastrointestinal (GI) tract. Milkoligosaccharides found in various mammals differ greatly, andcomposition in humans is unique (Hamosh M, 2001 Pediatr Clin North Am,48:69-86; Newburg D, 2001 Adv Exp Med Biol, 501:3-10). Moreover, glycanlevels in human milk change throughout lactation and also vary widelyamong individuals (Morrow A et al., 2004 J Pediatr, 145:297-303;Chaturvedi P et al., 2001 Glycobiology, 11:365-372). Approximately 200distinct human milk oligosaccharides have been identified andcombinations of simple epitopes are responsible for this diversity(Newburg D, 1999 Curr Med Chem, 6:117-127; Ninonuevo M et al., 2006 JAgric Food Chem, 54:7471-74801). Human milk oligosaccharides arecomposed of 5 monosaccharides: D-glucose (Glc), D-galactose (Gal),N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetylneuraminic acid, Neu5Ac, NANA). Human milk oligosaccharides are usuallydivided into two groups according to their chemical structures: neutralcompounds containing Glc, Gal, GlcNAc, and Fuc, linked to a lactose(Galβ1-4Glc) core, and acidic compounds including the same sugars, andoften the same core structures, plus NANA (Charlwood J, et al. 1999 AnalBiochem, 273:261-277; Martin-Sosa, et al. 2003 J Dairy Sci, 86:52-59;Parkkinen J and Finne J, 1987 Methods Enzymol, 138:289-300; Shen Z, etal. 2001 J Chromatogr A, 921:315-321).

Approximately 70-80% of oligosaccharides in human milk, e.g.,2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL),lactodifucotetraose (LDFT), are fucosylated. Secretor fucosylglycans inhuman milk and secretor fucosylated epitopes in the infant gut selectfor microbiota that interact with fucose. These microbiota promotehealthy colonization, and thereby reduce infections and inflammatorydiseases of the gastrointestinal tract.

Human milk glycans have structural homology to cell receptors forenteropathogens and function as receptor decoys. For example, pathogenicstrains of Campylobacter bind specifically to glycans containing H-2,i.e., 2′-fucosyl-N-acetyllactosamine or 2′-fucosyllactose (2′-FL).Campylobacter binding and infectivity are inhibited by 2′-FL and otherglycans containing this H-2 epitope. Similarly, some diarrheagenic E.coli pathogens are strongly inhibited in vivo by human milkoligosaccharides containing 2-linked fucose moieties. Several majorstrains of human caliciviruses, especially the noroviruses, also bind to2-linked fucosylated glycans, and this binding is inhibited by humanmilk 2-linked fucosylated glycans. Consumption of human milk that hashigh levels of these 2-linked fucosyloligosaccharides was associatedwith lower risk of norovirus, Campylobacter, ST of E. coli-associateddiarrhea, and moderate-to-severe diarrhea of all causes in a Mexicancohort of breastfeeding children (Newburg D et al., 2004 Glycobiology,14:253-263; Newburg D et al., 1998 Lancet, 351:1160-1164).

Human Milk Glycans as Prebiotics

Human milk contains a complex oligosaccharide mixture with prebioticcharacteristics. As described herein, human milk oligosaccharides suchas 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), andlactodifucotetraose (LDFT) have prebiotic properties and areadministered individually or in combination to stimulate the growth ofspecific beneficial bacteria that modulate mucosal immunity and conferprotection against pathogens in newborns. Prebiotic oligosaccharides areshort-chain carbohydrates with a degree of polymerization between 2 and60, and are indigestible by human or animal digestive systems. Thedefining property of prebiotics is their ability to selectivelystimulate the growth of beneficial bacteria including bifidobacteria andlactobacilli in the large intestine (Cummings J H and Macfarlane G T,2002 Br J Nutr, 87(suppl 2):S145-S151). The prebiotic oligosaccharidesare fermented by the gut flora, resulting in the release of hydrogen andcarbon dioxide gas and short-chain fatty acids such as butyrate. Theshort-chain fatty acids reduce the pH of the stools, which leads to amild laxative effect with softening and increased frequency of stools.Thus, prebiotics, including the purified 2′-FL, 3-FL and, LDFT describedherein prevent the constipation that is frequently observed informula-fed infants. In addition, the acidic pH prevents growth ofpathogens, promotes robust growth of healthy organisms, and promotesintegrity of colonic epithelial cells.

Prebiotics encourage selective changes in both the composition andactivity of the gastrointestinal microbiota, thereby conferring manyhealth benefits upon the host, including the reduction of enteric andother diseases. Prebiotics stimulate the growth of the beneficialbacteria including bifidobacteria, the genus often predominant in theintestinal microbiota of breast-fed infants. The prebiotic HMOSdescribed herein arrive undigested in the distal gut and act assubstrates for fermentation.

As described herein, human milk glycans function as prebiotic agentsthat stimulate beneficial colonization of the infant gut. For example,in 1905, a unique predominance of lactobacilli, particularlyLactobacillus bifidus (now classified as Bifidobacterium bifidum) wasnoted in the microflora of breastfed infants. In 1953, a ‘bifidusfactor’ was isolated from human milk that specifically stimulated growthof this bacterium. The milk factor was characterized in 1974 as a glycancontaining N-acetylglucosamine, galactose, glucose, fucose, and sialicacid; however, sialic acid removal did not reduce activity. The amountof growth-promoting glycan activity varied widely among milk fromdifferent individuals. Bovine milk and infant formula contained an orderof magnitude less ‘bifidus’ activity than human milk.

As described herein, ingestion of specific purified human milk glycans,e.g., 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL) andlactodifucotetraose (LDFT) cause specific beneficial bacteria/symbiontsto dominate the infant microbiome. This is associated with positiveconsequences to the microbial ecosystem of the neonatal gut andsubsequent health of the child. However, the prebiotic HMOS describedherein are not limited to use in human infants. The prebiotic HMOS ofthe invention are administered to an infant or adult mammal. The mammalcan be, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, adog, a cat, a horse, as well as livestock or animals grown for foodconsumption, e.g., cattle, sheep, pigs, chickens, and goats. Preferably,the mammal is a human.

Prior to the invention described herein, the addition of mixtures offructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) toinfant formula stimulated colonization by B. bifidum and severallactobacilli (See also, Bakker-Zierikzee A, et al. 2005 British Journalof Nutrition, 94, 783-790). Described in detail below is the ability ofmilk glycans, such as 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL)and lactodifucotetraose (LDFT) to promote colonization by beneficialmicrobes. Although bacteria have been isolated from the human microbiomethat utilize human milk oligosaccharides (Marcobal A, et al. 2010 JAgric Food Chem, 58(9): 5334-5340; Ward R E, et al. 2006 Appl EnvironMicrobiol, 72(6): 4497-4499; Ward R E, et al. 2007 Mol Nutr Food Res,51(11): 1398-1405), prior to the invention described herein, theprebiotic effect of individual, purified human milk oligosaccharides,either native or synthetic, was unknown.

Purified human milk oligosaccharides 2′-FL (MW=488), 3-FL (MW=488) andLDFT (MW=635) are utilized as prebiotics to promote the proliferationand residence of beneficial bacteria in the human intestine. Asdescribed in detail below, the prebiotic effects of human milkoligosaccharides including 2′-fucosyllactose were analyzed on themicrobial community of infants, and on individual microbes isolated fromhuman microbiota known to utilize human milk oligosaccharides. Thetesting was performed on anaerobic cultures of these communities andisolates of the microbiota.

Secretor Human Milk Glycans and Secretor Infant Gut Glycans PromoteColonization by Secretor-Binding Microbiota

Secretor fucosylglycans in human milk and secretor fucosylated epitopesin the infant gut select for microbiota that interact with fucose. Thesemicrobiota promote healthy colonization, and thereby reduce infectionsand inflammatory diseases of the gastrointestinal tract. The bifidusfactor described above is only one minor component of HMOS. As describedbelow, the fucosyloligosaccharides represent approximately 85% of thehuman milk glycome, and also stimulate the growth of specific microbesto produce a beneficial pattern of microbiota. Fucosylated glycans arealso prevalent on the mucosal cell surface as the gut matures. Commonmembers of the human microbiota, including Bacteroides fragilis,Bacteroides thetaiotaomicron, and Roseburia inulinivorans, utilizefucose during their colonization of the intestine. As described above,these three phenomena are related, and constitute an integrated systemfor introduction of specific types of microbes into the intestinalmicrobial community.

Expression of glycans in the intestine changes over the course of infantdevelopment, and glycan content in human milk changes over the course oflactation. These systems work synergistically to direct microbialsuccession of the gut microbiota toward the more complex pattern seen inadults. Gut glycan expression varies among individuals in relation totheir histo-blood group types, and expression of fucosyl glycans in milkreflects variation in maternal Lewis histo-blood group type. Thus, inthe gastrointestinal (GI) tract of the breastfeeding infant, variationin glycans provided by milk and variation in the glycans expressed oninfant mucosal surface combine to produce specific glycan patterns thatsupport specific combinations of microbiota. These patterns and degreesof microbiota colonization are determinants of specific outcomes,including risk of necrotizing enterocolitis.

As described in detail below, the plant prebiotic fructooligosaccharidestimulated growth, reduced pH, and induced the production of lactic acidin fecal slurrys containing the human microbiome and individual bacteriathat are part of the human microbiome. Human milk oligosaccharides(HMOS) also exhibit these prebiotic traits. As described below,individual pure, synthetic HMOS 2′-FL, 3-FL and LDFT promote bacterialgrowth, decrease pH, and induce the production of lactic acid. Prior tothe invention described herein, purified 2′-FL, 3-FL and LDFT were notindividually characterized as prebiotic compounds. As described below,prebiotic agents such as 2′-FL, 3-FL and LDFT are associated with manyhealth benefits, including decreased enteric and other infectiousdiseases, decreased constipation, increased calcium absorption, anddecreased risk of inflammatory disease.

Due to the surge in disease in infants at weaning, the HMOS of theinvention are added to weaning foods (e.g., mashed rice, bananas,porridges and other gruels, formula, etc.) to reduce or ameliorate thesediseases. Optionally, the HMOS are added to the weaning foods during themanufacturing process. Alternatively, the synthetic HMOS are added tothe weaning foods after the manufacturing process, but prior toingestion. For example, packets of sugars including HMOS are added toweaning foods prior to infant ingestion.

EXAMPLE 1 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL) andlactodifucotetraose (LDFT) are prebiotic agents

The hypothesis that human milk oligosaccharides as a group, and that theindividual human milk oligosaccharides 2′-fucosyllactosel,3-fucosyllactose and lactodifucotetraose are prebiotic was testeddirectly on mixed microbial communities derived from infant feces, andon purified microbial strains isolated from human microbiota. Testingwas performed on anaerobic cultures of these communities and isolates ofthe microbiota.

First, the impact of 2′-FL and HMO were examined on fresh samples ofmixed microbiota of three infants. A carbohydrate-free basal medium wasprepared according to Hughes (Hughes S, et al. 2008 FEMS Microbiol Ecol,64(3): 482-93) to study the growth of fecal bacteria during fermentationof human milk oligosaccharides (HMO) or 2′-FL. This basal mediumcontained per liter: 2 g peptone (Oxoid Ltd., Basingstoke, UK), 2 gyeast extract (Oxoid), 0.1 g NaCl, 0.04 g K₂HPO₄, 0.01 g MgSO₄.7H₂O,0.01 g CaCl₂.6H₂O, 2 g NaHCO₃, 0.005 g haemin (Sigma), 0.5 g L-cysteineHCl (Sigma), 0.5 g bile salts (Oxoid), 2 ml Tween 80, 10 μl vitamin K(Sigma), and 4 ml of 0.025% (w/v) resazurin solution. Anaerobic culturemethods were those described in Bryant (Bryant, M 1972 Am J Clin Nutr,25(12): 1324-8) using Hungate culture tubes, sealed with butyl rubbersepta. Media were prepared and maintained anaerobically using O₂-freeCO₂.

Fresh fecal samples were collected from three healthy babies, identifiedas A, B and C, who had not received antibiotics or pre/probiotics andhad no history of gastrointestinal disorder. Fresh human infant feceswere added to phosphate-buffered sterile anaerobic saline solution (1:10feces to saline), mixed in a blender for 60 s, homogenized and filteredthrough a double layer of sterile cheesecloth. 2 g/L 2′FL and 5 g/L HMOwere dissolved for 1 hour in medium before inoculation with 10% (v/v)fecal slurry prepared as above. Hungate tubes containing 2′-FL or HMOwere inoculated with fecal material, serving as the treatment. Hungatetubes containing no substrate were also inoculated with feces, to serveas negative control. All tubes were incubated at 37° C. Culturesupernatant samples were taken at 24 h. All samples were stored at −20°C. for further analysis. FIGS. 1, 2 and 3 show the results. Prebioticsare associated with an increase in the numbers of microbiota, especiallybifidobacteria and lactobacilli, a decrease in gut pH, and an increasein lactic acid. FIG. 1 demonstrates that both 2′-FL at 2 g/L and HMOS at5 g/L induced an increase in total bacteria in microbiota derived fromthe fecal slurry of 3 individual infants after culture in vitro. FIG. 2demonstrates that pH is reduced in each of these three microbialcommunities after growth in the presence of 2′-FL or HMOS. Also, whenthese three microbial communities are each cultured in the presence of2′-FL or HMOS, significantly higher amounts of lactic acid are producedcompared to controls (FIG. 3). These changes are all characteristic ofprebiotics.

Next, the responses to human milk oligosaccharides of a number ofpurifed bacterial strains that had mainly been isolated from human(infant) feces were examined. Individual bacterial isolates (mostlybifidobacteria and a few bacteroidetes) were tested in the presence orabsence of 2′-FL at 2 g/L, 3-FL at 2 g/L, LDFT at 2 g/L and HMOS at 5g/L, approximating the average concentrations of these sugars in humanmilk FOS (fructooligosaccharide, a prototypic plant prebiotic) at 2 g/Lwas also tested (approximating the concentrations of FOS used in infantformulae). Bacterial strain isolates (Table 1) were obtained from theJapanese Collection of Microorganism (RIKEN BioResource Center, Japan),and the American Type Culture Collection (Manassas, Va.).

TABLE 1 Species Origin Bifidobacterium longum JCM 7007 Feces of humaninfant Bifidobacterium longum JCM 7009 Feces of human infantBifidobacterium longum JCM 7010 Feces of human infant Bifidobacteriumlongum JCM 7011 Feces of human infant Bifidobacterium longum JCM 1210Feces of human infant Bifidobacterium longum JCM 1260 Feces of humaninfant Bifidobacterium longum JCM 1272 Feces of human infantBifidobacterium longum JCM 11347 Feces of human infant Bifidobacteriumlongum ATCC 15708 Feces of human infant Bifidobacterium longum ATCC15697 Feces of human infant Bacteroides vulgatus ATCC 8482 n/aBacteroides fragilis ATCC 25285 Appendix abscess Clostridium perfringensATCC 13124 n/a Escherichia coli ATCC 35401 Human feces

For these experiments a chemically defined medium (ZMB1) containingamino acids but no carbohydrates, and which supports the growth of manyenteric bacteria was used (Zhang, G., Mills, D. A., and Block, D. E.(2009) Appl Environ Microbiol 75, 1080-1087). For the individual straingrowth experiments, ZMB1 media supplemented with either 2 g/L 2′-FL, 2g/L 3-FL, 2 g/L LDFT or 5 g/L HMO were prepared. Each bacterial culturewas then innoculated to 0.5 OD (600 nm) at the outset. ZMB1 culturescontaining no additional oligosaccharide substrate served as negativecontrols., while ZMB1 cultures containing 2 g/L FOS served as positivecontrols. All bacteria were grown in anaerobic conditions at 37° C.,using an anaerobic chamber (DG250 Anaerobic Workstation, Don WhitleyScientific Limited, West Yorkshire, UK). Culture supernatant sampleswere taken at 48 h. Growth was assessed by optical density (OD 600 nm)in a microtiter plate. Culture pH was also recorded after 48 h ofgrowth. Lactic acid concentrations in the cultures at the end offermentation were assayed using a lactate assay kit (kit no. K607-100;BioVision Inc., CA, USA). All experiments were carried out inquadruplicate.

For most of these bacteria, growth was stimulated (to various degrees)by 2′-FL, 3-FL, LDFT, HMOS or FOS, and the pH of cultures decreased(FIGS. 4 and 5). In most cases the decrease in pH was as at least asgreat for 2′-FL, 3-FL and LDFT as for FOS, which is an exemplaroligosaccharide with prebiotic activity. These data indicate thatpurified 2′-FL, 3-FL and LDFT each can function as a highly potentprebiotic independent of other HMOS components.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed:
 1. A method for stimulating the growth of anexogenously administered probiotic bacterium in a gastrointestinal tractof a mammalian subject, comprising: administering to said subject acomposition comprising an oligosaccharide, wherein said oligosaccharideconsists of (a) purified 2′-fucosyllactose (2′-FL), (b) purified3-fucosyllactose (3-FL), (c) purified lactodifucotetraose (LDFT), or (d)a combination thereof, wherein the composition further comprises aprobiotic bacterium selected from the group consisting ofBifidobacterium bifidum, Bacteroides fragilis, Bacteroidesthetaiotaomicron, Lactobacillus acidophilus, Lactobacillus rhamnosus,Bacteroides vulgatus, Lactococcus lactis, and Streptococcusthermophilus, wherein said 2′-FL, said 3-FL, or said LDFT is at least95%, 98%, or 99% pure, and wherein said oligosaccharide selectivelystimulates the growth of said probiotic bacterium.
 2. The method ofclaim 1, wherein the local pH in said gastrointestinal tract isdecreased following administration of said composition.
 3. The method ofclaim 1, wherein the local lactate or lactic acid concentration in saidgastrointestinal tract is increased following administration of saidcomposition.
 4. The method of claim 1, wherein the growth of apathogenic bacterium in said gastrointestinal tract is inhibitedfollowing administration of said composition.
 5. The method of claim 1,wherein said composition comprises between 0.01 g 2′ FL and 1 g 2′-FLper gram of composition.
 6. The method of claim 4, wherein saidpathogenic bacterium is selected from the group consisting ofClostridium histolyticum, Clostridium difficile, Clostridiumperfringens, Enterobacter aerogenes, Listeria monocytogenes,Staphylococcus aureus, Salmonella enterica, Yersinia enterocolitica,enterotoxigenic E. coli, and enteropathogenic E. coli.
 7. The method ofclaim 1, wherein said subject is an infant.
 8. The method of claim 1,wherein said composition is in the form of an infant formula.
 9. Themethod of claim 1, wherein said composition is provided in dry milk, inmashed rice, in a banana, in a porridge, or in a gruel.
 10. A method forstimulating the growth of an exogenously administered probioticbacterium in a gastrointestinal tract of a mammalian subject,comprising: administering to said subject a composition comprising (a)purified 2′-fucosyllactose (2′-FL), (b) purified 3-fucosyllactose(3-FL), (c) purified lactodifucotetraose (LDFT), or (d) a combinationthereof, wherein said 2′-FL, said 3-FL, or said LDFT is at least 95%,98%, or 99% pure, wherein said oligosaccharide selectively stimulatesthe growth of said probiotic bacterium and wherein the exogenouslyadministered probiotic bacterium comprises a probiotic bacteriumselected from the group consisting of Bifidobacterium bifidum,Bacteroides fragilis, Bacteroides thetaiotaomicron, Lactobacillusacidophilus, Lactobacillus rhamnosus, Bacteroides vulgatus, Lactococcuslactis, and Streptococcus thermophilus.
 11. A method for stimulating thegrowth of an exogenously administered probiotic bacterium in agastrointestinal tract of a mammalian subject, comprising: administeringto said subject a composition comprising 2′-fucosyllactose (2′-FL), aprobiotic bacterium selected from the group consisting ofBifidobacterium bifidum, Bacteroides fragilis, Bacteroidesthetaiotaomicron, Lactobacillus acidophilus, Lactobacillus rhamnosus,Bacteroides vulgatus, Lactococcus lactis, and Streptococcusthermophilus, and a pharmaceutically-acceptable excipient or inactiveingredient, wherein said 2′-FL is at least 95%, 98%, or 99% pure, andwherein said 2′-FL selectively stimulates the growth of said probioticbacterium.
 12. The method of claim 1, wherein said probiotic bacteriumis selected from the group consisting of Bifidobacterium bifidum BB-12,Lactobacillus acidophilus RC-14, Lactobacillus acidophilus NCFM,Lactobacillus acidophilus CL 1285, Lactobacillus rhamnosus GG, andLactobacillus rhamnosus GR-1.
 13. The method of claim 10, wherein saidprobiotic bacterium is selected from the group consisting ofBifidobacterium bifidum BB-12, Lactobacillus acidophilus RC-14,Lactobacillus acidophilus NCFM, Lactobacillus acidophilus CL 1285,Lactobacillus rhamnosus GG, and Lactobacillus rhamnosus GR-1.
 14. Themethod of claim 1, wherein said probiotic bacterium further comprisesBifidobacterium infantis or Bifidobacterium longum.
 15. The method ofclaim 1, wherein said composition comprises yogurt or a probioticbeverage.
 16. The method of claim 1, wherein said probiotic bacterium isBacteroides fragilis, Bacteroides thetaiotaomicron, or Bacteroidesvulgatus.
 17. The method of claim 1, wherein said composition comprisesa weaning food.
 18. The method of claim 1, wherein said mammaliansubject is not an infant.
 19. The method of claim 18, wherein saidmammalian subject is a child.
 20. The method of claim 18, wherein saidmammalian subject is an adult.
 21. The method of claim 6, wherein saidpathogenic bacterium is selected from the group consisting ofEnterobacter aerogenes, Listeria monocytogenes, Staphylococcus aureus,Salmonella enterica, and Yersinia enterocolitica.